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@mberacochea
mberacochea committed Jun 5, 2024
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24 changes: 17 additions & 7 deletions README.md
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Expand Up @@ -35,7 +35,7 @@
The workflow uses the following tools and databases:

| Tool/Database | Version | Purpose |
|--------------------------------------------------------------------------------------------------|-----------------------------------------------|------------------------------------------------------------------------------------------------------------------------|
| ------------------------------------------------------------------------------------------------ | --------------------------------------------- | ---------------------------------------------------------------------------------------------------------------------- |
| [Prokka](https://github.com/tseemann/prokka) | 1.14.6 | CDS calling and functional annotation (default) |
| [Bakta](https://github.com/oschwengers/bakta) | 1.9.3 | CDS calling and functional annotation (if --bakta flag is used) |
| [Bakta db](https://zenodo.org/record/10522951/) | 2024-01-19 with AMRFinderPlus DB 2024-01-31.1 | Bakta DB (when Bakta is used as the gene caller) |
Expand All @@ -56,7 +56,7 @@ The workflow uses the following tools and databases:
| [cmscan](http://eddylab.org/infernal/) | 1.1.5 | ncRNA predictions |
| [Rfam](https://rfam.org/) | 14.9 | Identification of SSU/LSU rRNA and other ncRNAs |
| [tRNAscan-SE](https://github.com/UCSC-LoweLab/tRNAscan-SE) | 2.0.9 | tRNA predictions |
| [pyCirclize](https://github.com/moshi4/pyCirclize) | 1.4.0 | Visualise the merged GFF file |
| [pyCirclize](https://github.com/moshi4/pyCirclize) | 1.4.0 | Visualise the merged GFF file |
| [VIRify](https://github.com/EBI-Metagenomics/emg-viral-pipeline) | 2.0.0 | Viral sequence annotation (runs separately) |
| [Mobilome annotation pipeline](https://github.com/EBI-Metagenomics/mobilome-annotation-pipeline) | 2.0 | Mobilome annotation (runs separately) |

Expand All @@ -79,7 +79,7 @@ Although it's possible to run the pipeline on a personal computer, due to the co
The pipeline needs reference databases in order to work, they take roughly 110G.

| Path | Size |
|---------------------|------|
| ------------------- | ---- |
| amrfinder | 217M |
| antismash | 9.4G |
| bakta | 71G |
Expand Down Expand Up @@ -123,6 +123,7 @@ maximum length is 24 characters;
`taxid` is the NCBI TaxId (if the species-level TaxId is not known, a TaxId for a higher taxonomic level can be used). If the taxonomy is known, look up the TaxID [here](https://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi).

#### Finding TaxIds

If TaxIds for input genomes are not known, a tool such as [CAT/BAT](https://github.com/MGXlab/CAT_pack) can be used.
Follow the [instructions](https://github.com/MGXlab/CAT_pack?tab=readme-ov-file#installation) for getting the tool and downloading the NCBI nr database for it.

Expand All @@ -137,6 +138,7 @@ CAT add_names -i BAT_results/${genome_name}.bin2classification.txt -o BAT_result
```

To generate an input file for `mettannotator`, use [generate_input_file.py](preprocessing/generate_input_file.py):

```
python3 preprocessing/generate_input_file.py -h
usage: generate_input_file.py [-h] -i INFILE -d INPUT_DIR -b BAT_DIR -o OUTFILE [--no-prefix]
Expand All @@ -160,16 +162,17 @@ optional arguments:
--no-prefix Skip prefix generation and leave the first column of the output file empty for
the user to fill out. Defaule: False
```

For example:

```bash
python3 generate_input_file.py -i list_of_genome_fasta_files.txt -d /path/to/the/fasta/files/folder/ -b BAT_results/ -o mettannotator_input.csv
```

It is always best to check the outputs to ensure the results are as expected. Correct any wrongly detected taxa before starting `mettannotator`.

Note, that by default the script uses FASTA file names as prefixes and truncates them to 24 characters if they exceed the limit.



### Running mettannotator

Running `mettannotator` with the `--help` option will pull the repository and display the help message:
Expand Down Expand Up @@ -258,6 +261,7 @@ nextflow run ebi-metagenomics/mettannotator \
> see [docs](https://nf-co.re/usage/configuration#custom-configuration-files).
### Gene caller choice

By default, `mettannotator` uses Prokka to identify protein-coding genes. Users can choose to use Bakta instead by
running `mettannotator` with the `--bakta` flag. `mettannotator` runs Bakta without ncRNA and CRISPR
annotation as these are produced by separate tools in the pipeline. Archaeal genomes will continue to be annotated using
Expand Down Expand Up @@ -343,13 +347,12 @@ Additionally, for genomes with no more than 50 annotated contigs, a Circos plot

<img src="media/circos-plot-example.png">


#### Data sources

Below is an explanation of how each field in column 3 and 9 of the final GFF file is populated. In most cases, information is taken as is from the reporting tool's output.

| Feature (column 3) | Attribute Name (column 9) | Reporting Tool | Description |
| --------------------- | ----------------------------------------------------------------------- |-----------------| ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- |
| --------------------- | ----------------------------------------------------------------------- | --------------- | ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- |
| ncRNA | all\* | cmscan + Rfam | ncRNA annotation (excluding tRNA) |
| tRNA | all\* | tRNAscan-SE | tRNA annotation |
| LeftFLANK, RightFLANK | all\* | CRISPRCasFinder | CRISPR array flanking sequence |
Expand Down Expand Up @@ -413,14 +416,17 @@ The mobilome annotation workflow is not currently integrated into `mettannotator
After installing both tools, follow these steps to add the mobilome annotation:

1. Run the [viral annotation pipeline](https://github.com/EBI-Metagenomics/emg-viral-pipeline):

```bash
nextflow run \
emg-viral-pipeline/virify.nf \
-profile <profile> \
--fasta <genome_fasta.fna> \
--output <prefix>
```

2. Run the [mobilome annotation pipeline](https://github.com/EBI-Metagenomics/mobilome-annotation-pipeline):

```bash
nextflow run mobilome-annotation-pipeline/main.nf \
--assembly <genome_fasta.fna> \
Expand All @@ -434,7 +440,9 @@ nextflow run mobilome-annotation-pipeline/main.nf \
--skip_amr true \
-profile <profile>"
```
3. Integrate the output into the `mettannotator` GFF
```bash
# Add mobilome to the merged GFF produced by mettannotator
python3 postprocessing/add_mobilome_to_gff.py \
Expand All @@ -448,7 +456,9 @@ python3 postprocessing/add_mobilome_to_gff.py \
-i <mettannotator_results_folder>/<prefix>/functional_annotation/merged_gff/<prefix>_annotations_with_descriptions.gff \
-o <prefix>_annotations_with_descriptions_with_mobilome.gff
```
4. Optional: regenerate the Circos plot with the mobilome track added
```bash
pip install pycirclize
pip install matplotlib
Expand Down
84 changes: 37 additions & 47 deletions modules.json
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@@ -1,50 +1,40 @@
{
"name": "ebi-metagenomics/mettannotator",
"homePage": "https://github.com/ebi-metagenomics/mettannotator",
"repos": {
"https://github.com/nf-core/modules.git": {
"modules": {
"nf-core": {
"bakta/bakta": {
"branch": "master",
"git_sha": "9d0f89b445e1f5b2fb30476f4be9a8b519c07846",
"installed_by": [
"modules"
],
"patch": "modules/nf-core/bakta/bakta/bakta-bakta.diff"
},
"custom/dumpsoftwareversions": {
"branch": "master",
"git_sha": "05c280924b6c768d484c7c443dad5e605c4ff4b4",
"installed_by": [
"modules"
]
},
"gecco/run": {
"branch": "master",
"git_sha": "3f5420aa22e00bd030a2556dfdffc9e164ec0ec5",
"installed_by": [
"modules"
],
"patch": "modules/nf-core/gecco/run/gecco-run.diff"
},
"multiqc": {
"branch": "master",
"git_sha": "a6e11ac655e744f7ebc724be669dd568ffdc0e80",
"installed_by": [
"modules"
]
},
"quast": {
"branch": "master",
"git_sha": "344638191a5d6b3526556410819dfcf24e98039e",
"installed_by": [
"modules"
],
"patch": "modules/nf-core/quast/quast.diff"
}
}
}
"name": "ebi-metagenomics/mettannotator",
"homePage": "https://github.com/ebi-metagenomics/mettannotator",
"repos": {
"https://github.com/nf-core/modules.git": {
"modules": {
"nf-core": {
"bakta/bakta": {
"branch": "master",
"git_sha": "9d0f89b445e1f5b2fb30476f4be9a8b519c07846",
"installed_by": ["modules"],
"patch": "modules/nf-core/bakta/bakta/bakta-bakta.diff"
},
"custom/dumpsoftwareversions": {
"branch": "master",
"git_sha": "05c280924b6c768d484c7c443dad5e605c4ff4b4",
"installed_by": ["modules"]
},
"gecco/run": {
"branch": "master",
"git_sha": "3f5420aa22e00bd030a2556dfdffc9e164ec0ec5",
"installed_by": ["modules"],
"patch": "modules/nf-core/gecco/run/gecco-run.diff"
},
"multiqc": {
"branch": "master",
"git_sha": "a6e11ac655e744f7ebc724be669dd568ffdc0e80",
"installed_by": ["modules"]
},
"quast": {
"branch": "master",
"git_sha": "344638191a5d6b3526556410819dfcf24e98039e",
"installed_by": ["modules"],
"patch": "modules/nf-core/quast/quast.diff"
}
}
}
}
}
}
}
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